POWRE: The Structural and Energetic Basis of Transcriptional Control in E. coli pap Operon

Previous work has led to a model for regulation known as the phase variation control mechanism, in which the methylation state of two GATC sequences determines the binding sites for the dimeric leucine-responsive regulatory protein. Methylation of upstream GATC-I results in Lrp binding to promoter-proximal sites, steric blockage of RNA polymerase and transcriptional repression. If GATC-I becomes umethylated Lrp dimers translocate to upstream sites mediated by the coregulator protein PapI and GATC-II is methylated and transcription is activated. The experiments in this grant will provide a structural and quantitative foundation to evaluate and further explore this phase variation model. Thermodynamic dissociation constants will be measured by gel shift analysis for Lrp binding to wild-type and selected mutant sites, and the consequences of GATC methylation and Pap I binding will be explored. X-ray crystallography will determine the high resolution atomic structure of unliganded Lrp, Lrp-DNA complexes, and ternary Lrp-PapI-DNA complexes. These biochemical and X-ray crystallographic data will be analyzed and used to develop models for the structure of the DNA control region. This is a POWRE award which will allow the PI, who is currently a research associate, to establish and independent research project.